Bone Morphogenetic Protein 4 in the Extraembryonic Mesoderm Is

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Bone morphogenetic protein 4 in the extraembryonic mesoderm is required for allantois development and the localization and survival of primordial germ cells in the mouse

Takeshi Fujiwara*, N. Ray Dunn*†, and Brigid L. M. Hogan*‡§

*Department of Cell Biology, Vanderbilt University Medical Center, and ‡Howard Hughes Medical Institute, 1161 21st Avenue South, Nashville, TN 37232-2175

Communicated by R. L. Brinster, University of Pennsylvania, Philadelphia, PA, September 26, 2001 (received for review July 3, 2001) Evidence suggests that the specification of primordial germ cells a cumulative process, initiated by signaling between the tropho- (PGCs) in the mammalian embryo does not depend on maternal blast and the inner cell mass (ICM) of the blastocyst and determinants. Rather, previous genetic analysis in the mouse has continued between the ExE and the proximal epiblast. shown that bone morphogenetic protein 4 (Bmp4) is required for The idea that interactions between the ExE and epiblast are the formation of both PGCs and allantois. Bmp4 is expressed in required for PGC development is supported by experimental and both the trophoblast-derived extraembryonic ectoderm (ExE) and genetic evidence. For example, if epiblast cells of the prestreak in the epiblast-derived extraembryonic mesoderm (ExM), in which embryo are cultured in contact with the ExE, some of the the PGCs, allantois primordium, and angioblasts are first detected. epiblast cells differentiate into AP-positive, presumptive PGCs We have shown that Bmp4 made in the ExE functions to induce (4). Moreover, studies suggest that this interaction is mediated precursors of PGCs and allantois in the adjacent epiblast, resulting by extracellular signaling proteins, including bone morphoge- in complete lack of both cell types in homozygous null mutants. netic proteins (Bmps) Bmp4 and Bmp8b, both of which are However, the function of Bmp4 in the ExM is totally unknown. To expressed in the ExE. Thus, Bmp4 homozygous null mutants address this question, we generated tetraploid (4N) chimeras by completely lack PGCs and an allantois, whereas Bmp8b homozy- aggregating Bmp4 null ES cells with wild-type tetraploid embryos. gous null mutants have either a significantly reduced number of In this combination, wild-type tetraploid cells contribute to the PGCs or none at all, and poor development of the allantois (5–7). extraembryonic trophoblast and primitive endoderm lineages but Culture of distal epiblasts with cells expressing Bmp4 and Bmp8b are excluded from the epiblast and its derivatives, including the results in the appearance of AP-positive, presumptive PGCs ExM. Our results clearly demonstrate that Bmp4 made in the ExM after about 72 h (8). does not affect the establishment of either PGC or allantois In addition to its expression in the ExE, Bmp4 is also tran- lineages, but is required for PGC localization and survival and for scribed in the extraembryonic mesoderm (ExM). At the late the differentiation of the allantois. These findings suggest that streak stage, Bmp4-expressing cells are predominantly localized Bmp4 expressed in epiblast-derived tissues plays vital roles in at the periphery of the incipient allantois bud, abutting the reproduction by regulating both the development of the germ line exocoelom and separated from the cluster of PGCs by a popu- and the vascular connection between the embryo and the placenta. lation of non-Bmp4-expressing mesenchyme cells. By the headfold stage, Bmp4-expressing cells are more extensively distrib-

n the mouse embryo, primordial germ cells (PGCs) are first uted within the allantois, and some are seen adjacent to the BIOLOGY PGCs (ref. 5 and Fig. 4 B and C). Taken together, these results Idetected at the head-fold stage as a cluster of about 45 alkaline DEVELOPMENTAL phosphatase (AP)-expressing cells at the base of the allantois suggest that Bmp4 has the potential to affect PGC and allantois (1). These PGCs subsequently migrate into the endoderm that development at several different steps: first, in a dose-dependent gives rise to the hindgut (2), distribute along the hindgut, and way in the establishment of precursors in the epiblast, then in the finally move through the dorsal mesentery into the genital ridges allocation of these precursors to the PGC or allantois lineages, (reviewed in ref. 3). Lineage analysis has shown that the PGCs and finally in the survival and differentiation of these cell types are derived before gastrulation from a subpopulation of proxi- once they have been specified. mal epiblast cells adjacent to the trophoblast-derived extraem- In this paper, we test the last two hypotheses by performing lacZ/tm1 bryonic ectoderm (ExE). Significantly, these precursors are not tetraploid chimera analysis. Lines of Bmp4 null embryonic lineage restricted but have descendants in both the germ line and stem (ES) cells were established and aggregated with wild-type extraembryonic tissues, such as the allantois, yolk sac mesoderm, tetraploid (4N) morulae to generate chimeric embryos (9). With and amnion (1). this strategy, the trophoblast and ExE will express Bmp4 (and Previous studies suggest that the specification of the mouse Bmp8b), and the primitive endoderm-derived visceral endoderm germ line does not involve, as in many other organisms, the inheritance of maternal determinants. Rather, it appears that Abbreviations: Bmp, bone morphogenetic protein; PGC, primordial germ cell; ExE, ex- interactions between the trophoblast-derived ExE and the ad- traembryonic ectoderm; ExM, extraembryonic mesoderm; ES, embryonic stem; 4N, tet- jacent epiblast first establish precursors that can give rise to both raploid; AP, alkaline phosphatase; ICM, inner cell mass; VCAM-1, vascular cell adhesion PGCs and extraembryonic mesoderm cells. A second process molecule 1. then allocates descendants of these cells to the different lineages †Present address: Department of Molecular and Cellular Biology, Harvard University, (reviewed in ref. 3). This model says nothing about whether the Cambridge, MA 02138. interactions induce the de novo expression of genes specifically §To whom reprint requests should be addressed at: Department of Cell Biology, Vanderbilt University Medical Center, C-2310 MCN, Nashville, TN 37232-2175. E-mail: brigid.hogan@ required for PGC development, or whether they maintain in the mcmail.vanderbilt.edu. PGCs expression of key factors permitting pluripotency that are The publication costs of this article were defrayed in part by page charge payment. This normally lost in cells that differentiate into somatic lineages. article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Moreover, the induction of the PGC͞allantois precursors may be §1734 solely to indicate this fact.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.241508898 PNAS ͉ November 20, 2001 ͉ vol. 98 ͉ no. 24 ͉ 13739–13744 Downloaded by guest on September 30, 2021 will express Bmp2 (7), but the ICM derivatives, including the ExM, will completely lack Bmp4 expression. We find that these chimeric embryos do contain PGCs at the presomite stage, in about the same number as control chimeras. However, most of the PGCs are incorrectly localized in the yolk sac, from which they later disappear. Mutant tetraploid chimeras also show abnormal development of the allantois, which fails to extend and fuse with the chorion and lacks a normal vasculature. We conclude that Bmp4 expressed in the ExM is not required for the allocation of precursors into the PGC and allantois͞ExM lineages but does affect the subsequent behavior and survival of the PGCs and the differentiation of the allantois. Materials and Methods Excision of the Floxed neor Cassette in Bmp4lacZneo ES Cells and Generation of Transheterozygous Bmp4 Null ES Cells. 12C Bmp4lacZneo ES cells (50 ϫ 106) (5) were electroporated with 100 ␮g of cytomegalovirus (pCMV)-Cre plasmid. The neo excised clones were screened by Southern blot analysis of SpeI-digested genomic DNA with the 5Ј external 500-bp BsmI–BamHI Bmp4 genomic probe (10). One clone, 10G, was chosen for the generation of transheterozygous Bmp4 null ES cell lines. 10G ES cells (16.4 ϫ 106) were electroporated with 50 ␮gofNotI- linearized Vb replacement vector, which targets exons 1 and 2 of the Bmp4 allele with a neo (neomycin-resistance) gene (10). Clones were screened by Southern blot analysis of XbaI-digested genomic DNA with the 5Ј external Bmp4 genomic probe as described. Five of 142 clones were identified as Bmp4lacZ/tm1 transheterozygous null lines (data not shown). Two lines, 5H and 6H, were chosen for further investigation by chimeric analysis. The heterozygous Bmp4lacZ/ϩ line, 3E, was used as a control.

Generation of Tetraploid Chimeric Embryos. Tetraploid embryos were produced by aggregating four-cell stage tetraploid embryos with 12–16 ES cells as described (9).

␤-Galactosidase Staining and Detection of PGCs. ␤-Galactosidase staining, histological analysis, and whole-mount AP staining of tetraploid chimeric embryos were performed as described (5). Fig. 1. Morphology and posterior differentiation of tetraploid chimeras. Whole-Mount Immunostaining. Whole-mount immunostaining was (Left) Bmp4lacZ/ϩ chimeras. (Right) Bmp4lacZ/tm1 chimeras. (A and B) Lateral performed as described (11). Embryos were exposed to primary views of ﬁve-somite (5S; A) and four-somite (4S; B) tetraploid chimeras after staining with 5-bromo-4-chloro-3-indolyl ␤-D-galactoside (X-Gal) to reveal antibodies against Pecam1 (PharMingen), Flk1 (PharMingen), lacZ or vascular cell adhesion molecule 1 (VCAM-1) (PharMingen) Bmp4 expression. Both chimeras show expression in the allantois, amnion, lateral plate mesoderm, and heart. Note the well-formed, extended allantois at a dilution of 1:150. Horseradish peroxidase-conjugated don- in the control (A) and unextended allantois in the null mutant chimera (B). (C key anti-rat IgG (Jackson ImmunoResearch) was used as a and D) Section through posterior of seven- or eight-somite chimeras in a plane secondary antibody at a dilution of 1:1,000. Antibody binding similar to that indicated in A and B. Embryos were stained with X-Gal, and was detected with the use of diaminobenzidine hydrochloride sections were counterstained with eosin. Note the close proximity of the substrate. mutant allantois to the posterior of the embryo and the presence of some blood vessels within the tissue (D). No vitelline artery can be seen in this or Cell Proliferation Assay. Embryos were fixed and stained for adjacent sections of the mutant chimera. (E and F) Whole-mount in situ hybridization of one- or two-somite stage chimeras with Stoma probe. (G and ␤-galactosidase activity as described above. After fixation in 4% ͞ H) Reduced posterior primitive streak in mutant tetraploid chimera. Posterior paraformaldehyde PBSfor1hat4°C embryos were dehydrated view of two chimeras after whole-mount in situ hybridization with Fgf8 in 100% isopropyl alcohol, embedded in wax, and serially probe. Note that the null mutant chimera is at the six-somite stage, whereas sectioned at 7 ␮m. Anti-phosphohistone H3 antibody staining of the control chimera is at the three-somite stage. (I and J) Section through the serial sections was performed after antigen retrieval in 10 mM same chimera as in C and D. Note the abnormal morphology of the neural tube sodium citrate solution. Sections were exposed to the primary and somites in the mutant (J). da, Dorsal aorta; hg, hindgut; np, neural plate; nt, neural tube; s, somite; va, vitelline artery. (Scale bars: A, B, E, and F, 200 ␮m; antibody at 1:500 dilution in normal donkey serum overnight at C, D, G–J, 100 ␮m.) room temperature. After three 15-min changes of PBS they were exposed to secondary Cy3-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch) at 1:1,000 dilution for1hat4°C. After three 15-min changes of PBS, YO-PRO-1 (Molecular Results Probes) was used at 1:5,000 dilution in PBS for nuclear coun- Generation of Tetraploid Chimeras with Bmp4lacZ/tm1 Null ES Cell Lines. terstaining. Slides were mounted with 50% glycerol͞2% n-propyl Tetraploid chimeras were generated by aggregating Bmp4lacZ/tm1 gallate͞PBS, and the number of positive and negative nuclei in null ES cells (see Materials and Methods) with wild-type 4N ICR all sections was counted. embryos. At the early somite stage, Bmp4lacZ/tm1 7 wild-type

13740 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.241508898 Fujiwara et al. Downloaded by guest on September 30, 2021 expression pattern and is transcribed strongly in the early allantois (A. Shimono and R. Behringer, personal communi- cation). Strong localized expression of Stoma was observed in both Bmp4lacZ/ϩ and Bmp4lacZ/tm1 tetraploid chimeras, suggesting that mesoderm cells can be specified as allantois in the absence of Bmp4 expression in the ExM (Fig. 1 E and F). Moreover, expression of Fgf8 was used to assess primitive streak formation (12). Whole-mount in situ hybridization analysis revealed that in the Bmp4lacZ/tm1 tetraploid chimeras the primitive streak was indeed formed but was somewhat shorter than normal (Fig. 1 G and H). Finally, histological analysis of the mutant chimeras showed complete lack of the vitelline artery as well as aberrant development of the neural tube and somites (Fig. 1 I and J).

PGC Development in Bmp4lacZ/tm1 Tetraploid Chimeras. To test the role of Bmp4 made in the ExM on PGC development, chimeric embryos were subjected to whole-mount AP staining. At the presomite stage most of the PGCs in the four control Bmp4lacZ/ϩ tetraploid chimeras analyzed were detected in their normal location at the base of allantois. A few were seen in the yolk sac, but none were seen within the allantois (Fig. 2A and Table 1). At high magnification after AP staining, wild-type PGCs had a regular, rounded appearance (Fig. 2G). In contrast, in Bmp4lacZ/tm1 tetraploid chimeras at this stage most PGCs were found dispersed in ectopic sites (yolk sac, amnion, and allantois), Fig. 2. PGCs in tetraploid chimeras. Whole-mount AP staining of Bmp4lacZ/ϩ with fewer at the base of the allantois (Fig. 2 B and C and Table (A and E) and Bmp4lacZ/tm1 (B–D and F) chimeras. PGCs are indicated by 1). In one of the four mutant chimeras analyzed there was a large arrowheads. At the presomite stage in the control chimera (A) most PGCs are clump of presumptive PGCs inside the allantois (Fig. 2D), so it detected at or near the base of the allantois. One mutant chimera gave a was not possible to ascertain the total number of PGCs in this similar distribution (not shown). In two mutant chimeras, PGCs were more chimera. Significantly, the overall number of countable PGCs dispersed within the yolk sac, as shown in the posterior–lateral view in B.Ina did not differ between the two classes of chimeras. In the fourth chimera (C and D) PGCs were seen in both the posterior endoderm (C) Bmp4lacZ/ϩ tetraploid chimeras there were 37 Ϯ 6 PGCs (n ϭ 4), and a large clump of AP-positive cells in the allantois (D). At the four-somite whereas in the Bmp4lacZ/tm1 tetraploid chimeras there were 40 Ϯ stage, PGCs are detected in the forming hindgut pocket of control chimeras 5 PGCs (n ϭ 3). At high magnification, most PGCs, both at the (E), but only very few are seen in mutant chimeras (F). Insets in A–-C are higher magniﬁcations of the boxed areas. (G–I) Morphology of AP-stained PGCs in base of the allantois and in the yolk sac, had an irregular A–C. In the control chimera, PGCs at the base of the allantois had a regular appearance and numerous large cytoplasmic ‘‘blebs,’’ as might rounded morphology (G). In mutant chimeras, most PGCs within the yolk sac be expected if the cells were not adhering to the substratum or (H) and at the base of allantois (I) showed numerous large cytoplasmic blebs were about to undergo apoptosis (Fig. 2 H and I). (arrows). (Scale bars: A–-C, 200 ␮m; D–I, 100 ␮m.) At the two- to five-somite stage, a dramatic difference was seen between the two kinds of chimera in terms of PGC number. In the four Bmp4lacZ/ϩ tetraploid chimeras analyzed at this stage lacZ/tm1 BIOLOGY tetraploid chimeras (hereafter referred to as Bmp4 tet- most PGCs (38 Ϯ 5) were closely associated with the hindgut raploid chimeras) were somewhat smaller than control chimeras lacZ/tm1 DEVELOPMENTAL ϩ pocket. However, in the Bmp4 tetraploid chimeras, only (Fig. 1 A and B). In wild-type embryos and Bmp4lacZ/ tetraploid 12 Ϯ 8 PGCs (n ϭ 4) were observed in this location (Fig. 2 E and chimeras at this stage, the allantois has grown toward the chorion F and Table 1). The number of PGCs in the yolk sac was and has started to fuse with it. However, in 14 of the 16 significantly reduced to 3 Ϯ 3 from 21 Ϯ 10 in the presomite stage Bmp4lacZ/tm1 tetraploid chimeras analyzed here, the allantois was (Table 1). In the one Bmp4lacZ/tm1 tetraploid chimera analyzed at small and unextended and remained closely associated with the the seven-somite stage, no detectable PGCs were observed posterior of the embryo (Fig. 1 A–D). In the other two chimeras, (Table 1). Taken together, our findings suggest that Bmp4 the allantois was ballooned out but had not fused with the produced in the ExM is required for the proper localization and chorion (Fig. 3C, c and d). survival of PGCs once they are specified. To determine whether the allantoic mesoderm had been specified correctly, we attempted to determine whether the Abnormal Development of the Allantois in Mutant Chimeras. In the cells express Stoma. This gene normally has a very restricted normal embryo several processes contribute to the growth,

Table 1. Number of PGCs in respective somatic cells No. of PGCs in region

Stage Genotype Base of allantois Hindgut Yolk sac Allantois Amnion Overall

Presomite Bmp4lacZ/ϩ (4) 32 Ϯ 45Ϯ 31Ϯ 11Ϯ 137Ϯ 6 Bmp4lacZ/tm1 (3) 14 Ϯ 721Ϯ 10 2 Ϯ 2 (2) 2 Ϯ 240Ϯ 5 Two to ﬁve somites Bmp4lacZ/ϩ (4) 38 Ϯ 504Ϯ 2042Ϯ 5 Bmp4lacZ/tm1 (4) 12 Ϯ 83Ϯ 38Ϯ 71Ϯ 120Ϯ 8 Seven somites Bmp4lacZ/tm1 (1) 0 0 2 0 2

Results are presented as mean Ϯ SD. Numbers in parentheses are number of chimeric embryos examined.

Fujiwara et al. PNAS ͉ November 20, 2001 ͉ vol. 98 ͉ no. 24 ͉ 13741 Downloaded by guest on September 30, 2021 elongation, and differentiation of the allantois once the primordium of this tissue has been established (13–15). Initially, the allantois increases in size because of the recruitment of cells from the posterior primitive streak. By the two- to three-somite stage, cell proliferation and cavitation take over as the most important driving forces of expansion. At the same time, there is distal–proximal differentiation of Flk1-expressing angioblasts within the allantois and organization of endothelial cells into a network of Pecam1-positive blood vessels. Significantly, these latter events occur by local vasculogenesis, and not by invasion of the vitelline or fetal blood vessels from the embryo, and there is no accompanying erythropoiesis. Finally, first the distal, and then more proximal, cells of the allantois differentiate into a mesothelium expressing the gene encoding VCAM-1. To determine whether the morphological defect of the allantois of the mutant chimeras is due to reduced proliferation, we carried out immunohistochemistry with the use of anti- phosphohistone H3 antibody, which detects cells in G2 and mitosis. The ratio of phosphohistone H3-positive cells within the entire allantoic mesoderm was 5.0% Ϯ 0.7% in the Bmp4lacZ/tm1 tetraploid chimera compared with 4.6% Ϯ 0.7% in the control (Fig. 3A, a–d), suggesting that there was no significant difference in the proliferation rates of the two tissues. To examine vascularization in the mutant chimeras, we performed whole-mount immunohistochemistry with antibodies to Flk1 and Pecam1. Within the yolk sac and lateral plate mesoderm of both Bmp4lacZ/tm1 and Bmp4lacZ/ϩ tetraploid chimeras, there was little difference in the level of Flk1-positive cells (Fig. 3B, a and b, and data not shown), indicating that mesoderm can differentiate into angioblasts, despite the lack of Bmp4 expression in the ExM. In contrast, in the allantois of the Bmp4lacZ/tm1 tetraploid chimeras, the level of Flk1-positive cells was reduced compared with controls (Fig. 3B, a and b). Moreover, the number and complexity of blood vessels were also dramatically reduced in the allantois region, as revealed by Pecam1 staining (Fig. 3B, c–f). Two of the 16 Bmp4lacZ/tm1 tetraploid chimeras analyzed had an abnormal balloon-shaped allantois that failed to fuse with the chorion (Fig. 3C, c and d). This phenotype resembled that of embryos homozygous null for either VCAM-1 or ␣4 integrin, genes that encode surface molecules mediating specific adhesion between expressing cells (16–18). In mouse embryos from embryonic day 7.5 to 9.5, VCAM-1 is normally expressed in the distal mesothelial cells of the allantois that mediate chorioallan- toic fusion by interacting with ␣4 integrin-expressing cells in the chorion and in some internal mesenchyme cells (19). Whole- mount immunohistochemistry with an anti-VCAM-1 antibody revealed protein at the distal end of the allantois in the Bmp4lacZ/ϩ tetraploid chimera (Fig. 3C, a and b). However, staining was not seen in Bmp4lacZ/tm1 chimera (Fig. 3C, c and d), suggesting that Bmp4 signaling is required for VCAM-1 expression in the developing allantois. Discussion Fig. 3. Proliferation, vascularization, and mesothelium formation in the allantois in tetraploid chimeras. (A) Cell proliferation in the allantois. Sections Role of Bmp4 in Establishing the Germ Cell Lineage. Previous analysis are through the posterior of four-somite-stage chimeras, in a plane similar to of Bmp4 null mutant embryos showed that they completely lack that indicated in Fig. 1 A and B.(Left) Bmp4lacZ/ϩ chimeras. (Right) Bmp4lacZ/tm1 both PGCs and an allantois, whereas Bmp8b null embryos have chimeras. (a and b) Immunofluorescence staining with antibody to phospho- few or no PGCs and a small allantois (5–8). Because Bmp4 and histone H3. (c and d) Counterstaining with Yo-Pro-1 to reveal all nuclei. (B) Vascularization of the allantois. (Left) Bmp4lacZ/ϩ chimeras. (Right) Bmp4lacZ/tm1 chimeras. (a and b) Whole-mount immunostaining of headfold (HF)-stage chimeras with antibody to Flk1. Note the lower number of positive head) in f.(C) Differentiation of the mesothelium: (Upper) Bmp4lacZ/ϩ chimera cells (arrows) in the allantois of the mutant tetraploid chimera (b) compared at seven-somite stage and (Bottom) Bmp4lacZ/tm1 chimera at nine-somite stage, with control (a). No significant difference was seen in the lateral plate (ar- both after whole-mount immunostaining with antibody to VCAM-1. (b) rowheads). (c–f) Whole-mount immunostaining of seven-somite- (c and e) and Higher magnification of chimera in a, showing VCAM-1 expression (arrows) in five-somite- (d and f) stage chimeras with antibody to Pecam1. The allantois the distal cells of the allantois. (d) Higher magnification of the chimera in c, of the control chimera (c) shows a complex network of blood vessels (arrow), showing the absence of VCAM-1 expression in the distal cells of the balloon- illustrated at higher power magnification in e. In contrast, the mutant allan- like allantois. al, Allantois; da, dorsal aorta; nt, neural tube. [Scale bars: A tois (d and f) has few vessels (arrows). Note the kinked neural tube (arrow- (a–d), B (e and f), and C(band d), 100 ␮m; B (a–d) and C (a and c), 200 ␮m.]

13742 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.241508898 Fujiwara et al. Downloaded by guest on September 30, 2021 Fig. 4. Model for the role of Bmp signaling in early PGC development in the mouse. (A) Before gastrulation. Bmp4 and Bmp8b, produced together in the extraembryonic ectoderm (ExE), and Bmp2 in the visceral endoderm (VE) function together to induce precursors of PGCs and extraembryonic mesoderm (ExM) in the proximal epiblast. At this stage epiblast cells are not lineage restricted, and single cells, denoted here by red triangles with inner circles, can give rise to descendants in the PGCs, allantois, and ExM. Other cells (blue triangles) give rise to descendants in the allantois and ExM, but not PGCs. Evidence suggests that Bmp4 and Bmp8b act on the epiblast cells through different receptors (8), and Smad5 also plays a role at this stage (25). (B) Late streak-incipient allantois bud stage: high magniﬁcation of the posterior region boxed in A, excluding the primitive streak. By this stage the allocation of cells to the PGC (red circles) and somatic cell (red triangle) lineages has taken place. Bmp4 expression is seen in mesoderm cells adjacent to the exocoelom but not immediately around the PGCs (5). (C) Headfold stage. Somatic cells within the allantois express Bmp4 (dark blue) and Bmp2 (light blue). PGCs are no longer in a tight clump and move (red stars) to the endoderm of the future hindgut (2). Bmp4 may affect PGC migration and survival directly, or indirectly by affecting the differentiation of the ExM and͞or the endoderm (blue arrows). In the absence of Bmp4 or Smad5 (25), PGCs may be swept passively into ectopic locations by expansion of the allantois and yolk sac or may no longer be prevented from moving there.

Bmp8b are coexpressed in the ExE before gastrulation it was somite stage are not localized at the base of allantois but are proposed that the two signaling proteins function in a dose- dispersed throughout the yolk sac, amnion, and allantois. In one dependent manner to establish PGC͞allantois precursors in the case, PGCs may possibly have aggregated into a large clump proximal epiblast. This hypothesis was supported by the finding within the allantois (Table 1). This mislocalization may be the that wild-type ES cells are unable to rescue the PGC and result of defects extrinsic to the PGCs or defects in the PGCs allantois phenotype when combined with Bmp4tm1 Ϫ͞Ϫ em- themselves (or both). For example, the mesenchyme cells around bryos, either by blastocyst injection or by morula aggregation (5). the PGCs or the presumptive hindgut endoderm with which they The reciprocal tetraploid chimera experiments described here later associate (2, 3) may require Bmp4 to differentiate correctly are also consistent with the hypothesis that the formation of PGC and to produce the chemoattractants, chemorepellants, or ex- precursors in the proximal epiblast absolutely requires Bmp4 tracellular matrix factors that normally either promote or restrict ͞ made by the trophoblast ExE and does not, as suggested by PGC migration and behavior. Alternatively, Bmp4 in the ExM others (8), depend upon any Bmp4 expression in ICM cells at the may directly or indirectly affect the differentiation of the PGCs, blastocyst stage (20). This interpretation assumes that in the and their expression of receptors for extracellular matrix pro- BIOLOGY chimeras the host wild-type 4N embryos never contribute any teins or adhesion molecules, or cellular machinery needed for DEVELOPMENTAL cells to the ICM of the blastocyst. However, it could be argued response to guidance cues in the environment. The abnormal that a few 4N cells are present transiently in the ICM before morphology of PGCs in the mutant chimeras supports the idea being competed out by 2N cells, and that they produce enough that these cells are not interacting normally with their environ- Bmp4 protein to maintain a subpopulation of ICM cells con- ment and may be undergoing a high rate of apoptosis. For taining cell autonomous factors permissive for the pluripotent whatever reason, if the PGCs are unable to actively move into the state. However, we consider this possibility unlikely, for two hindgut endoderm, they may be passively swept into the yolk sac reasons. First, genetic evidence suggests that Bmp4 functions in or allantois, as argued by others (2, 21). a dose-dependent way in establishing PGCs. Because the number By the early somite stage, the overall number of PGCs in of transient 4N cells in the ICM would likely be small, the lacZ/tm1 alternative model would predict significantly fewer founder Bmp4 tetraploid chimeras has declined, relative to earlier PGCs in mutant chimeras than in controls, which was not stages, to control chimeras. The reduction is seen in both ectopic observed. Second, our previous chimera experiments showed and normal locations, suggesting that the loss, presumably by that a high proportion of wild-type ES cells, which presumably apoptosis (Fig. 2 H and I), is not just due to PGCs being in an would make Bmp4 in the ICM, failed to rescue PGC formation abnormal location. The apparent failure of PGCs in the hindgut in Bmp4 Ϫ͞Ϫ embryos (5). pocket to survive may be due to defects in the local environment The fact that both founder PGCs and allantois cells are present or to the fact that, in the absence of Bmp4, PGCs do not express in Bmp4lacZ/tm1 tetraploid chimeras rules out the possibility that receptors for survival factors. A number of candidates have been Bmp4 plays a role in the allocation of common precursor cells to identified as survival factors for early PGCs in vivo, including either germ-line or somatic lineages. Other factors as yet un- Fgfs, stem cell factor, and members of the IL-6͞LIF cytokine known are therefore likely to control this decision. family (3). We have shown here that Fgf8 is still expressed in the posterior of Bmp4lacZ/tm1 tetraploid chimeras, but we have not yet Bmp4 Made by Extraembryonic Mesoderm Affects PGC Localization investigated the expression of other potential PGC survival and Survival. Unlike the PGCs in control chimeras, most of the factors or the possibility that Bmp4 itself functions as a survival PGCs present in Bmp4lacZ/tm1 tetraploid chimeras at the pre- factor in the ExM.

Fujiwara et al. PNAS ͉ November 20, 2001 ͉ vol. 98 ͉ no. 24 ͉ 13743 Downloaded by guest on September 30, 2021 Requirement of Bmp4 for the Growth and Differentiation of the Smad1, -5, -8, and -4 (reviewed in ref. 22). Genes encoding these Allantois. In all Bmp4lacZ/tm1 tetraploid chimeras examined an proteins have widespread expression at the presomite and early allantois-like structure is formed that expresses at least one somite stages (23, 24). Recent studies have shown that Smad1 allantois marker. However, the allantois is always abnormal in null mutants have few PGCs and allantois defects, albeit less size and morphology and fails to fuse with the chorion. The severe than described here (25). In Smad5 null mutants the absence of Bmp4 expression in the ExM has no significant effect founding population of PGCs is significantly smaller than nor- on the overall proliferation rate of allantois cells. However, at the mal, and they become ectopically localized in the ExM, as presomite stage, mutant chimeras have a marked deficiency of described here. The differentiation of the allantois and amnion both Flk1-positive angioblasts and Pecam1-expressing blood is also abnormal, suggesting defects in mesodermal lineage ͞ vessels. This deficiency suggests that locally produced Bmp4 allocation and or cell behavior within the ExM (21). These protein plays two roles in allantois vasculogenesis, the first in results are consistent with overlapping functions for both Smad1 promoting differentiation of allantoic mesoderm cells into an- and Smad5 in the induction of PGC precursors in the epiblast gioblasts and͞or in the survival of these cells once they have and the differentiation of ExM cells and PGCs once they are formed, and the second in promoting the assembly of endothelial formed, but not in the actual allocation of precursors to the germ cells into organized blood vessels. Finally, the absence of cell or somatic lineages. The model outlined in Fig. 4 has yet to VCAM-1 expression may be due to a failure in the initial be completed by identification of the Bmp receptors involved formation of an outer layer of mesothelium in the absence of and the downstream target genes of Bmp signaling in each cell Bmp4 expression in the allantoic mesoderm, or to a defect in the population. differentiation of any mesothelial cells that are present. We thank Andras Nagy and Marina Gertsenstein for precious and informative advice on making chimeras. We thank Kirstie Lawson for Potential Signaling Components Mediating the Effect of Bmp4 on critically reading the manuscript. We also thank Angela D. Land- PGCs and the Allantois. Implicit in the discussion above is that cells Dedrick for assistance in manuscript preparation. T.F. was supported by of the allantois and ExM, and possibly also the PGCs, express a grant from the National Institutes of Health (HD 28955) to B.L.M.H., Bmp4 receptors and downstream signaling components such as who is an Investigator of the Howard Hughes Medical Institute.

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